Cloning of genes in bacterial systems has greatly expanded the scope of molecular biology and gene therapy. Many genes, however, have not yet been successfully cloned or sub-cloned by conventional cloning techniques due to their toxicity in host cells such as bacteria. For example, cloning and sub-cloning of env genes from HIV-1 has been particularly difficult due to the toxicity of env genes in bacteria. Neither directional cloning by two enzyme digestion nor unidirectional cloning by single enzyme digestion or blunt-end ligation has proven to be successful for env genes or portions thereof which are toxic in bacteria.
Several approaches have been examined for the ability to clone toxic genes in bacteria For example, using a cytomegalovirus promoter in bacteria has not been successful because the promoter is leaky and the resultant protein is toxic to the bacteria. A possible solution is to choose a promoter that is not leaky. However, due to the paucity of non-leaky promoters and promoters which are as strong as the cytomegalovirus promoter, this strategy has not proven to be very successful. Another possible solution is to choose a strain of bacteria that is not as vulnerable to the toxic gene. Several strains including DH5α, XL-1 Blue, Top10F′, InvαF′, Sure, and HB101 have been used without much success.
Another possible strategy for cloning toxic genes in bacteria is to use antisense technology to bind to the mRNA encoding the toxic gene. For example, bacteria could be co-transformed with two plasmids—one encoding the toxic gene and a second plasmid encoding the antisense strand, which is lost once the selective pressure, such as ampicillin, is removed. Results based on such a strategy, however, have not been successful possibly due to the incompatibility of the ori of the two plasmids or possible due to the harshness of the double selection of the competent cells. A disadvantage of this strategy is that two plasmids carrying a long identical insert would likely recombine.
Accordingly, there remains a need for cloning toxic genes in bacteria. The present invention provides a single plasmid that can be used to clone the toxic gene in bacteria yet allow expression in mammalian cells. The present invention also provides methods of cloning toxic genes in bacteria using the plasmids.